Viroid quasispecies revealed by deep sequencing

Abstract

Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than 5 £ 10 ¡ 3 , quite comparable to the value of 2:5 £ 10 ¡ 3 reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

Mutational Analysis of Eggplant Latent Viroid RNA Circularization by the Eggplant tRNA Ligase in Escherichia coli

“…Then the viroid progeny moves cell-to-cell through the plasmodesmata, the channels that traverse the cell walls of plant cells, and long-distance through the phloem to establish a systemic infection in the host plant frequently inducing a disease. The more than 30 viroid species known to date are classified into two families, Pospiviroidae and Avsunviroidae , depending on the presence of particular domains in the viroid molecule; more specifically a central conserved region (CCR) that is present in all members of the family Pospiviroidae , but is missing in those of the Avsunviroidae , and hammerhead ribozymes that are exclusive of this last family ( Flores et al, 2015 ; Daròs, 2016b ; Steger and Perreault, 2016 ; Brass et al, 2017 ; Giguère and Perreault, 2017 ).…”

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

“…These replicative intermediates are then cleaved into (+) monomers by RNAse III [ 21 ] and circularized by DNA ligase I accepting RNA substrates [ 22 ] ( Fig 1 ). Although analysis of genetic diversity suggested differences in replication fidelity between nuclear and chloroplastic viroids [ 9 – 11 ], recent work based on previously published deep sequencing data has posited that potato spindle tuber viroid (PSTVd), the type species of the family Pospiviroidae , might show extremely high copying error rates similar to those of CChMVd [ 23 ]. It therefore remains to be elucidated whether the two viroid families show different rates of spontaneous mutation.…”

Viroid research and its significance for RNA technology and basic biochemistry

“…The error rates of chrysanthemum chlorotic mottle viroid ( 66 ) and eggplant latent viroid ( 67 ), two members of Avsunviroidae , were found to be in the range of 1/1000 to 1/400, while the error rate of PSTVd seems to be lower by a factor of ∼5 ( 67 , 68 ). The high error rate observed fits well into the theory of the viral quasispecies developed by Eigen ( 69 ) and of the inverse correlation between genome size and replication fidelity ( 70 , 71 ).…”